Localization of the gam gene of bacteriophage Mu and characterisation of the gene product

Gene, 49 (1986) 273-282

273

Elsevier GEN 01855

Localization of the gam gene of bacteriophage Mu and characterisation (Recombinant DNA; Mu gam linear DNA)

gene;

of the gene product

plasmid and Ml3 cloning; DNA binding protein; transformation

with

J. Akroyd” and N. Symonds b* a Imperial Cancer Research Laboratories. St. Bartholomew’s Hospital, Dominion House, Bartholomew b Close, London ECIA 7BE (U.K.) Tel. (01)7264708, and b School of Biological Sciences, University of Sussex, Brighton, BNl 9QG, Sussex (U.K.) Tel. (0273)606755 (Received July 15th, 1986) (Accepted October 6th, 1986)

SUMMARY

Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome. An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection agaist exonuclease action (Gam function). It also stimulates the frequency with which linear plasmid DNA transforms Escherikhia coli to antibiotic resistance (Sot function). The preliminary results reported here suggest that pgam is potentially a useful ‘tool’ in molecular biology, although the molecular details of pgam activity require further clarification.

INTRODUCTION

The temperate mutator phage Mu replicates its genome during the lytic growth cycle by multiple rounds of genetic transposition. The phage encoded genes A and B which are essential for efficient transposition, are located at the immunity or LHE region of the Mu genome and are expressed early during the * To whom correspondence

and reprint requests should be

addressed. Abbreviations: Ap, ampicillin; ATP, adenosine 5’-triphosphate; bp, base pair(s); BSA, bovine serum albumin; ccc, covalently closed circular; CIAP, calf intestinal alkaline phosphatase; Cm, chloramphenicol; ds, double-stranded; D’IT, ditiothreitol; IPTG, isopropyl-P-D-thiogalactosidase; kb, kilobase or 1000 0378-l 119/86/$03.50

0

1986 Elsevier

Science Publishers

B.V. (Biomedical

phage life-cycle (Fig. 1). The 5-kb stretch of DNA lying immediately to the right of the B gene is called the SE region of Mu. It is transcribed on the same early messenger as the A and B genes and from mini-cell experiments is known to encode at least nine proteins of 7-22 kDa (Magazin et al., 1978; Giphart-Gassler et al., 1981). The actual role of the SE region in Mu development is not known at bp; Km, kanamycin; LB, Luria Broth; LHE, left-hand end; nt, nucleotide(s); pgum, gam protein; POPOP, 1,4-bis(5-phenyl2-oxazolyl)benzene; PPO, 2,5diphenyloxazole; R, resistance; RF, replicative form; s, sensitivity; SE, semi-essential; Sm, Streptomycin; Tc, tetracycline; TCA, trichloroacetic acid; TY, see section b ofMATERIALS AND METHODS; ::, novel joint; A, deletion. Division)

214

RePressar+~~a~~a~~~‘_Head Genes Genes

Genes---+-----Tail

Genes +A

\

1 5kb

,

Fig. 1. Schematic map of the Mu genome. The continuous line represents the Mu genome (37 kb) and the broken line indicates the Mu DNA present in the strain PP258, described in MATERIALS AND METHODS, sections a and 1.

present

but a number of phenotypic effects have been traced to it and various ‘genes’ have been postulated to account for these effects (see Fig. 1). So far, the order of the putative genes on the Mu genome has not been determined and the M,s of the individual gene products are not known. It is in fact unclear whether various phenotypic effects are the result of separate genes or are different manifestations of a single gene product (see Goosen et al., 1982, for a review). Initially we have focussed our attention on two of the postulated genes, gum and sot whose phenotypes are connected in that they both relate to the protection of DNA from exonuclease attack. The gum gene of Mu is so called because it complements the gam gene of phage 1, a gene known to encode an inhibitor of exonuclease V (Van Vliet et al,, 1978). Expression of the sot gene in competent recipient cells stimulates the frequency of transfection with linear Mu DNA extracted from phage particles by any of the standard methods. The original hypothesis concerning sot was that in normal phage infection the injected virion DNA has Sot attached to its ends, thus protecting the Mu genome from exonuclease attack. The Sot would be lost during DNA purilication, and so for successful transfection to occur it would have to be already present in recipient cells (Van de Putte et al., 1977). We have previously reported the cloning of the gam and sot genes on a multicopy plasmid under the I pL promoter, controlled by the temperaturesensitive repressor encoded by ~1857 (Akroyd et al., 1985). Preliminary experiments indicated that the gam and sot genes were both present on a 1.6-kb fragment of DNA located between 5.1 and 6.7 kb from the LHE of Mu. When the proteins synthesised

from a plasmid clone carrying this fragment of DNA were analysed using both the mini- and maxi-cell techniques, a single protein of approximately 20 kDa was identified as specified by the Mu DNA (Akroyd, 1985). This result strongly suggests that the Gam and Sot phenotypes are different manifestations of a single gene, and in the following we shall assume this is so and refer to the gene as gum and its product as pram. In this paper we first describe the subcloning of the Mu DNA from pJA21 into the RF of the phage M 13, which in conjunction with an in vitro assay for pgam activity has allowed the gam gene to be precisely mapped on the Mu genome. We also present a preliminary analysis of the properties of pgam as deduced from in vivo and in vitro experiments in which high levels of pgam are obtained from induced cells carrying the pJA21 plasmid.

MATERIALS AND METHODS

(a) Bacteria, plasmids and phage (I) Bacterial strains

E. coli strains used were: JMlOld (luc-pro) thi supE [F’traD36, proAB + luclqZdMlS] (Messing et al., 1981); CSH26/1 arud(lac-pro) toti Su- thi (Miller, 1972); PP258 recA tip: : Mutts MAD-& rspL Sue (Goosen et al., 1982). (2) Plasmids Plasmids included: pBR322 and pMB9 (Bolivar et al., 1977); R300B (Bagdasarian et al., 1979); pSU1 (Maynard Smith et al., 1980); and pPH126

215

(Spratt et al., 1986). Plasmid pXY228 was obtained from Dr. P. Highfield, The Wellcome Foundation. It is a multicopy plasmid carrying an ApR gene and the I pL promoter next to a ~1857 repressor gene. Plasmid pJA21 (Akroyd et al., 1985) carries the Mu gam gene on a 1.58-kb EcoRI fragment downstream from the Apt promoter in pXY228. Expression of thegum gene from pJA2 1 does not occur at 32 *C but can be induced by a temperature shift above 37°C which inactivates the cI857-coded repressor. (3) Phage Phages included Mucts62 (Howe, 1973) and M13mp9 (Messing and Vieira, 1982). The Ml3 clones M13mpJA-El and M13mpJA-E3 were constructed by subcloning the 1.6-kb EcoRI Mu fragment from pJA21 into the RF of M13mp9. Phage M13mpJA-E3 carries the Mu fragment in the Mutranscribed orientation as determined by restriction analysis of RF DNA, and is phenotypically Gam + as determined by the in vitro pgam assay; phage M13mpJA-El carries the fragment in the reverse orientation and is phenotypically Gam-. Phages M13mpJA-EALl, M13mpJA-EAL2, M13mpJAEASl and M13mpJA-EAS2 were constructed by subcloning the Mu EcoRI-AccI restriction fragments from pJA21 into M13mp9, as described in RESULTS AND DISCUSSION, SeCtiOn a. (b) Enzymes and media Restriction endonucleases, T4 DNA ligase, T4 DNA polymerase and calf intestinal alkaline phosphatase (CIAP) were obtained from Boehringer Mannheim and were used according to the manufacturer’s specifications. Bacterial strains other than JMlOl were grown in LB and plated on LB-agar containing Ap or Km at a final concentration of 100 pg/ml where appropriate, and Tc or Sm at a final concentration of 10 yg/ml for plasmid selection. Strain JMlOl was maintained on minimal medium containing thiamine at 2 ,ug/ml; liquid cultures were grown in 2 x TY medium (16 g tryptone, 10 g yeast extract, 5 g NaCl in 1 1 of distilled water) and plated on 2 x TY agar.

(c) Subcloning the Mu DNA from pJA21 into M13mp9 The 1,6-kb EcoRI Mu fragment from pJA21 was purified by elution of the restricted DNA from a gel after agarose gel electrophoresis, by the method of Drelzen et al. (1981). The purified fragment was restricted with AccI endonuclease and cloned into the RF of M13mp9 DNA obtained from Amersham Inte~ation~. Preliminary nt sequencing experiments (Akroyd, unpublished results) indicated that both the AccI cleavage sites in the Mu fragment have the recognition sequence GTATAC, which is different from the Ace1 cloning site in M13mp9, GTCGAC. Therefore the Mu EcoRI-AccI restriction fragments were made flush-ended using T4 DNA polymerase and cloned into the unique SmaI site on M13mp9. The orientations of the Mu insertions were determined by the hybridisation assay described in the next section, and were then confirmed by restriction analysis of RF DNA. (d) Hybridisation assay to determine the orientation of Mu DNA insertions in the single-stranded form of Ml3 phage The M13mp9 clones M13mpJA-El (Gain) and M13mpJA-E3 (Gam’ ), which carry the Mu gam gene on an EcoRI fragment in a defined orientation, were used to determine the orientation of subclones of the EcuRI fragment in M 13mp9. Single-stranded M13mpJA-El or Ml3mpJA-E3 DNA (0.1 g) was mixed with 0.1 pg single-stranded DNA of the subclone in 10 ~1hybridisation buffer (100 mM Tris, pH 8.5,lOO mM MgCl,) and incubated at 60°C for l-2 h. The mixture was then analysed by electrophoresis through 0.5% agarose. The mobility through the gel of any two DNA species which hybridised together and therefore carried Mu insertions of opposite polarity, was clearly retarded in comparison with an unhyb~dised control. Phage hybridising to Ml3mpJA-El (Gam-) but not to M13mpJA-E3 (Gam +) were concluded to be carrying insertions in the Mu-transcribed orientation. This conclusion was confuned by restriction analysis of RF DNA and by the pgam assay.

216 RESULTS AND DISCUSSION

(a) Localisation of the Mu gam gene The construction of the plasmid pJA21 (Fig. 2), which carries the Mu Gam and Sot functions on a 1.5%kb fragment of DNA expressed from the i pL promoter has already been described (Akroyd et al., 1985). The position of the the gum gene on the Mu genome was located more accurately by sub-cloning the two Mu EcoRI-AccI restriction fragments from pJA21 (Fig. 2) into the RF of M13mp9 (Messing and Vieira, 1982) and assaying them for Gam activity. The orientations of the Mu insertions were determined by hybridisation of the single-stranded forms of the phage to the recombinant M13mp9 phage standards M 13mpJA-E3 and M 13mpJA-El which respectively carry the EcoRI fragment from pJA2 1 in the Mu transcribed direction, or the reverse. Phage were isolated carrying each of the Mu EcoRI-AccI fragments from pJA21 in the Mu-tranorientation : M 13mpJA-EALl and scribed M13mpJA-EAL2 carry the 930-bp EcoRI-AccI restriction fragment, while M13mpJA-EAS 1 and M13mpJA-EAS2 carry the 530-bp fragment. The phage were tested for Gam activity. The results in

Table I clearly indicate that phage carrying the 930-bp Mu EcoRI-AccI fragment expressed Gam activity and that this activity was present whether or not IPTG was added to relieve repression of the lac operator region on M 13mp9 by the E. coli lad4 gene product expressed from the host bacterium JMlOl (Messing et al., 1981), indicating that the high level of luc repressor was titrated out by the many copies of M13mp9 carrying the luc operator region. On the other hand, phage carrying the 530-bp EcoRI-AccI fragment did not express a significant level of Gam activity. It was therefore concluded that the Mu gam gene lies within 930 bp immediately to the right of the AccI site which is located 5.75 kb from the LHE of Mu. These results contradict earlier reports that the gum gene spans (or maps immediately to the right of) the EcoRI site 5.1 kb from the LHE of Mu and codes for a 14-kDa protein (Giphart-Gassler and Van de Putte, 1979; Giphart-Gassler et al., 1981). It is likely that these figures refer to a gene immediately to the left of gam whose function is unknown. The exact location of the structural gum gene has subsequently been pin-pointed precisely by nucleotide sequencing of the Ml3 clones. The results show that the gam gene lies between 5801 and 6322 kb from the LHE of Mu and encodes a protein of 174 amino acids (Akroyd et al., 1986). (h) Characterisation of pgam: DNA binding activity

I

1 cI057

pJA21 L

9.28kb

1

Fig. 2. Restriction map of pJA21 showing the EcoRI and AccI cleavage sites. The heavy line represents the vector (pXY228) DNA, while the thin line represents Mu DNA on which the gam gene (displaying both the Gam and Sot functions) is located. Distances between the restriction sites are given in kb. The exact length of the 116-bp fragment is known from nt sequences (J.E.A., unpublished results) while the sizes of the other fragments were determined by restriction analysis. The left-most EcoRI site on the diagram corresponds to the site 5.1 kb from the LHE of the Mu genome. A, AccI; E, EcoRI; Ap, Apn gene.

A number of properties of pgam have been studied using cells harbouring pJA21 to synthesize high levels of the protein. Experiments have already been reported which show that in crude cell extracts pgam inhibits the action of exonuclease on linear ds Mu DNA by binding to the DNA and not by interacting with the exonuclease, and that pgum can effectively halt exonuclease attack even after the exonuclease has ‘invaded’ linear ds molecules (Akroyd et al., 1985). Using an in vitro assay for Gam activity (see footnotes to Tables I and II) we tested the action of pgam on a variety of linear tritiated ds plasmid substrates with either flush ends or with staggered 3’ or 5’ 4-nt protrusions generated by restriction enzymes. The results in Table II indicate that all these molecules are substrates for Gam activity, as well as linear plasmid DNA that has been treated with CIAP to remove the terminal 5’-phosphate groups. These results suggest that pgum is a non-

211

TABLE I The pgam activity from Mu/M13mp9 subclones a Phage tested

Mu fragment sub-b cloned from pJA21

% Degradation

Percent of

of2pg Mu [3H]DNA

pgam activity”

+ IPTG

- IPTG

64.0

65.0

0

9.0

89

M 13mp9 (control)

None

M 13mpJA-E3

Entire 1.6-kb Mu EcoRI fragment from pJA21

7.0

M 13mpJA-EALl M 13mpJA-EAL2 1

930-bp Mu EcoRI-AccI fragment from pJA21

7.4

7.9

88

8.0

8.3

88

M13mpJA-EASl M 13mpJA-EAS2 1

530-bp Mu EcoRI-AccI fragment from pJA21

50.0

51.0

22

63.0

62.0

2

a Phage M13mp9 and its recombinants were grown in JMlOl cells in both the presence and absence of IPTG. IPTG is a gratuitous inducer of the fuc operon and its presence induces the expression of genes cloned under the Zucpromoter in M13mp9, in this case pgam. Crude cell-free extracts to be assayed for pgam activity were prepared by Brij-lysis, as described by Barbour and Clarke (1970). Reaction mixtures in 100 ~1 buffer (5 mM MgCl,, 20 mM NaCI, 1 mM DTT, 100 pg BSA/ml, 200 PM ATP), included 2 pg linear 3H-labelled Mu DNA and 100 pg total protein, as determined by the Bradford protein assay (Bradford, 1976), from the cell-free extract to be assayed. The reaction was incubated at 37°C for 30 min and terminated by the addition of 100 ~1 of calf-thymus DNA (1 mg/ml) and 200 ~1 ice-cold 10% TCA. The acid-precipitated DNA was removed by a IO-min centrimgation at 4°C in an Eppendorfcentrifuge, and the total supernatant was assayed for acid-soluble counts in aqueous scintillant (100 ml toluene, 90 ml T&on-X 100,l g PPO, 18 mg POPOP). The presence of pgam in the extract protects the linear 3H-labelled DNA from exonuclease degradation.The results are of a single experiment. However, quantitatively similar results were obtained in two other experiments. ’ All fragments were cloned in the Mu-transcribed orientation as determined by restriction analysis. c The pgam activity is expressed relative to the M13mp9 control:

% pgam activity =

lOO(A - B), where A

A = % degradation of 2 pg Mu r3H]DNA (in the presence of IPTG) in cell extracts containing M13mp9, as a control. B = y0 degradation of 2 pg Mu [ 3H]DNA (in the presence of IPTG) in cell extracts containing the phage recombinants to be assayed.

specific DNA-binding protein which interacts with linear ds DNA molecules having either flush or staggered ends, and furthermore that it does not require a terminal phosphate group for binding. (c) Stimulation of the transformation efficiency of linear plasmid DNA by pgum Normally the level of transformation obtained using linearised plasmid DNA is 100 to 1000-fold less than the value found with an equivalent amount of the circular plasmid DNA (Conley and Saunders, 1984). One reason for this discrepancy is almost certain to be the susceptibility of linear DNA to exonuclease action. It therefore seemed of interest to

determine whether the discrepancy would be reduced if the competent cells used in the transformation assay contained the non-specific DNA-binding protein pgam. Initially experiments were performed using the recA - strain PP258 (Goosen et al., 1982), which carries the defective Mutts kil- gam + prophage (Fig. l), as the recipient for transformation, rather than a strain harbouring the plasmid pJA21, because the absence of any resident plasmid in PP258 facilitated restriction analysis of the plasmids present in the transformant colonies. The linear plasmid DNA used in the transformation experiments was pBR322 (Bolivar et al., 1977) restricted with either BamHI endonuclease to yield a linear molecule with complementary 4-nt 5’-extensions at

278 TABLE II Inhibition of degradation of a variety of DNA substrates by the gum/sot gene product” 3H-labelled plasmid substrate

Enzyme used to restrict substrate

Nature of the ends of the linearised molecules produced by restriction digestion

y0 Degradation of 2 pg of the linearised [‘HI substrate, incubated in an extract of MM294 recA cells carrying pJA21 (i) Induced

Percent of pgem activity

(ii) Non-induced

lOO(ii- i)/ii

BamHI

4-nt 5’-extension

7

66

89

SphI EcoRV

4-nt 3’-extension blunt

10 8

72 71

86 89

pBR322

BamHI 6t calf intestinal alkaline phosphatase

dephosphorylated 4-nt 3 ’ -extension

17

60

72

psu1 (approx. 50 kb)

SmaI

blunt

12

70

83

pMB9 (approx. 5.5 kb)

EcoRI

4-nt 5’-extension

9

65

86

R300B (approx. 8.25 kb)

HpaI

blunt

25

86

71

pBR322 (approx. 4.4 kb)

a Reaction mixtures were set up and assayed as described in the legend to Table I, except that they contained 2 ng ‘H-labelled linear plasmid DNA and 100 ng total protein from a cell-free extract of CSH26/1 cells carrying the plasmid pJA21 from which the gam protein was expressed. The results are the means of three separate experiments.

each end, or with EcoRV endonuclease which produces a linear flush-ended molecule. Both these enzymes have unique cleavage sites in pBR322 within the TcR determinant. Transformants were first selected on Ap-agar (Table III) and then replicaplated onto Tc-agar (Table IV) in order to determine the proportion of the transforming plasmids with deletions extending away from the site of linearisation. The ratio of the number of transformants obtained with linear DNA to the number obtained with ccc DNA was taken as an index of the relative transformation efficiencies of the linear plasmid DNA in diierent recipients. This ratio standardises the effects of variability in competence levels obtained in different experiments. The results in Table III show that the expression of pgum in the recipient PP258 prior to transformation stimulates the transformation frequency with linear plasmid DNA approximately loo-fold, irrespective of

whether pBR322 is cut with BamHI or EcoRV. It must be noted however that expression of pgum also reduces the transformation frequency of ccc pBR322 DNA about IO-fold. The frequency of deletions extending into the TcR determinant of pBR322 in three transformation experiments is presented in Table IV. It can be seen that this frequency is 510% lower when pgum is expressed in the recipient cells, a result that was consistent in all similar experiments we have performed. The sizes of the deletions in several TcS plasmids obtained from transformation of both Gam’ and Gam- recipients were determined by restriction analysis (data not shown). The deletions, extending into both BumHI and EcoRV-linearised pBR322 DNA, ranged from less than 200 bp to 1.0 kb in plasmids recovered from the Gam + recipient, and from less than 200 bp to 1.8 kb in plasmids recovered from the Gam- recipient. The largest

279 TABLE III Transformation of PP258 to ampicillin resistance with linear pBR322 DNA” Recipient strain PP258

DNA used in tr~sformation pBR322 ccc (Cl

pBR322 linearised with BumHI

pBR322 ccc (C)

% L/C

pBR322 linearised with EcoRV

% L/C

CL)

tu Mean number of

Mean number of transformants/j_4g

transformantsj~g PP258 induced

3.8 x 104

5.9 x IO2

1.6

1.2 x IO4

6.2 x IO2

5.2

FP258 non-induced

2.0 x 103

2.9 X 10’

0.015

9.8 x la4

4.3 X 10r

0.044

Induced/ non-induced

107

118

* Competent cells of the recipient PP258 or of ceils c~ry~n8 pIA were prepared using the calcium chloride technique (Cohen et al., 1973). PP258 carries the Mu gam gene next to the Mutts repressor gene on a defective prophage (see Fig. 1) and expression of pgurm can be induced by a shift in temperature. Competent cells were prepared from duplicate cultures grown in LB to a density of2 x 10’ cells/ml at 28°C; one culture was then shifted to 42°C for 15 min to induce pgam expression and then returned to 28°C to grow for a further 15 min prior to the preparation of competent cells, while the non-induced culture was grown continuously at 28°C as a control. The linear plasmid DNA to be used in transformation experiments was prepared by restriction endonuclease cleavage of ccc DNA to produce linear molecules with either blunt (EC&V) or staggered (BarnHI) termini. The ccc DNA was incubated with the restriction enzyme for 16 b under optimal reaction ~nd~t~ons to ensure that close to 190% ofthe plasmid molecules were cleaved, and the reaction was monitored by agarose gel eiectrophoresis. The same batch of endonuclease was used in experiments that were to be compared. The DNA was heated to 65°C for 10 min (to ‘melt’ any end-annealed DNA which may be formed by hydrogen-boding between the complementary staggered termini generated by some restriction enzymes), and it was then used directly for tr~sfo~ation. DNA in 5-W pl TE (10 mM Tris; I mM EDTA; pH 7.9)was mixed with 100 ~1 ofcomp~tent cells, incubated on ice for 1 h and then ‘heat-shocked’ for 3 min at 32°C. The mixture was diluted in 3 ml ofLB and grown with aeration for 45-60 min to allow expression of plasmid-encoded genes. h appropriate dilution of the transformed cells was plated on Ap-agar and the plates were incubated at 32°C until colonies appeared (16-24 b). The colonies were rep&a-plated onto Te-agar to determine whether deletions had occurred during tr~sfo~atio~ (see Table IV). A parallel experiment was always performed with unrestricted ccc DNA. Plasmid DNA was prepared from transformant colonies and analysed by restriction analysis. The results are the means of three separate experiments.

defetion that will stih leave a viable plasmid is 2.6 kb ; the extent of deletions of less than 200 bp could not be determined from our analysis, Once it had been demonstrated that a s~m~Iation of~~sfo~a~on with linear plasmid DNA could be obt~~ in the fP258 recipient ~xpr~ss~~ the gam gene a similar series of experiments were conducts using the plasmid pJA21 as a source of p&am, and as the transforming DNA either an R300B derivative (obtained from Dr. P. Barth, ICI) encoding resistance to both Sm and Km, or a pBR322 derivative piasmid pPH126 (Spratt et al., 1986) in which the ApR gene has been replaced by a KmR gene. In these experiments the recipients were either

grower ~0nt~uousIy at 37°C {co~~tions of partial induction of pgam), or were heat-induced at 43°C for intervals ranging from 5 to 20 min. Variable results were obtained in these experiments. In two sets (one performed at 37”C, the other after 10 min ofindu~~on at43”C) a 1Wfold stimulation in transfo~ation was observed, and the characteristics of the transformants closely resembled those reported here using PP258 as the recipient. Nowever, in other experiments, relativeiy little enh~cement ~minimum 3-fold) of tr~sfo~ation. frequency was detected. It is possible that the role of pgam in st~ula~~g transformation with linear DNA is due entirely to its ability to protect the ends of the DNA from

280 TABLE IV Frequencies of deletions obtained on transformation with linear pBR322 DNA

PP258 induced

PP258 non-induced

Enzyme used to linearise pBR322 DNA

Total number of Apn transformants tested (X)

Total number of TcS transformants obtained (Y)

Percent of deletions b

Mean

BamHI

61 78 31

40 49 21

60 63 68

64

EcoRV

60 33 36

48 27 27

80 82 75

79

BamHI

21 39 27

15 30 20

71 77 74

74

EcoRV

47 28 35

39 26 27

83 93 77

84

% deletions

a The bacterial recipient PP258, which carries the Mu gum gene, was transformed to ApR with plasmid pBR322 DNA linearised at either the BamHI or EcoRV restriction site located within the TcR gene, as described in the legend to Table III. The transformant colonies selected on Ap-agar were replica-plated onto Tc-agar to determine whether or not deletions had occurred in the linear pBR322 DNA during transformation, resulting in ApR TcS transformant colonies. b The % deletions were calculated as the y0 of the ApR transformants that were TcS, i.e., 100 (Y/X).

exonuclease attack, the rejoining being done by host enzymes. However, the fact that the DNA ligase of E. coli does not normally mediate blunt-end ligation suggests that pgum also plays a direct part in the rejoining reaction. As the gum protein has now been purified (Akroyd et al., 1986) it should be possible to answer this question by performing in vitro experiments. Potentially pgum is a useful ‘tool’ in molecular biology for protecting linear DNA from degradation both in vivo and in vitro. Due to its non-specific binding activity it can presumably be used in both eukaryotic and prokaryotic systems, and is likely to be particularly valuable in systems where linear intermediates are short-lived, such as in vitro studies involving recombination, transcription and translation. (d) The effect of high Gam activity on cell viability

One possible reason for the variability observed in the transformation experiments when recipients carried the plasmid pJA21 was that excess production of pgam affected cell viability. The results of an

experiment designed to test this proposal are presented graphicahy in Fig. 3. It can be seen that the expression of pgam from pJA21 at 43 ’ C reduces cell survival lOO-to lOOO-foldin 15 min. The variation in the rates of cell death seen in the two experiments is analogous to the variation seen in the transformation experiments with linear DNA and may reflect differential sensitivity of the cells to killing by pgam at slightly different phases of cell growth. The effect of a lower level of pgum expression on cell viability was also investigated by growing the cells at 37°C. No direct killing effect was observed under these conditions but cell growth was retarded. In log phase of cell growth the doubling time of a culture carrying pJA21, grown in the presence of 100 pg Ap/ml, was 60 min, as opposed to 20 min for a control experiment with a culture of the same bacterial strain carrying the cloning vector pXY228. It is difficult to tell whether these effects of pgum on cell viability and cell growth play any role in the normal Mu life cycle as the intracellular level of pgum in the induced cells is abnormally high because it is being expressed on a multicopy plasmid from a

281

Time of Heat Induction (minutes) Fig. 3. Cell survival in the presence of pXY228, and pgam expressed from pJA21. Overnight cultures of CSH26/1 cells carrying either the gam clone, pJA2l (squares), or the cloning vector pXY228 (circfes) as a control, were diluted l/20 into LB containing 100 pg Ap/ml, and grown with aeration to a density of f-2 x 10s cells/ml at 30°C. The cultures were then shiRed to 42°C to initiate pgum expression. Samples were taken at different time intervals and viable cells assayed on Ap-agar. Su~~ng colony-foxing units are plotted as the percentage of survivors relative to the numbers of viable cells present before the temperature shift. The results of two separate experiments are plotted (open and filled symbols).

strong promoter; at 43°C almost 20% of the total cellular protein being produced is known to be pgam (Akroyd, 1985). (e) What is the role

of pgam

in the

Mu life-cycle?

A pertinent question, still to be answered, concerns the role of pgam in the Mu life cycle. The original idea was that pgam is packaged with the virion DNA, and upon infection was injected into host cells attached to the ends of the phage DNA, thus protecting the Mu genome from exonuclease attack (Van de Putte et al., 1977). The properties of pgam repOrted in RESULTS AND DISCUSSION sections b and c are consistent with this notion, but two complicating lines of evidence need to be considered. Firstly, it has been shown directly that there is a protein attached to the ends of virion DNA which can protect it from exonuclease attack and

possibfy mediate an intracellular transition from a linear to a circular form (Chase and Benzinger, 1982; Harshey and Bukhari, 1983; Purspurs et al., 1983); however, the size of this protein was 64-65 kDa, not the 19 kDa of pgappz.The second piece of evidence concerns a phage mutant, MuAp.5, isalated in our laboratory (Leach, 1980), in which approx. 1.1 kb of Mu DNA has been substituted by a 1.4-kb fragment from the transposon Tn3, which carries the /Glactamase gene. Electron microscope observation has located this deletion at 6.2 f 0.5 kb from the LHE of Mu (A. Resibois, personal communication) which overlaps the 5.8-6.3-kb span of the gam gene, and in vitro assays have confirmed that MuAp5 is ph~ot~i~~ly Cam- (Akroyd et al., 1985). Since MuAp5 is a viable phage, it seems unlikely that pgam exerts the function of protecting virion DNA from exonuclease attack after injection (for which the 64-kDa protein seems the natural candidate), but the assertion that pgam does normally exert some influence during the phage life cycle is supported by the findings that MuAp5 lysogenises more efliciently than wild-type Mu, and its latent period is appreciably longer (Akroyd et al., 1985). One possibility is that pgam is directly involved at the site of tr~sposition~ binding non-specially both to the donor and target sequences in the transposition reaction. The non-essential nature of the gam gene can then be explained by the presence of a host DNA-binding protein which can perform similar functions to pgam, but with a different efficiency.

(1) The Mu gam gene maps within a 93%bp sequence lying immediately to the right of an Ace1 site which is located 5.75 kb from the LHE end of the Mu genome. This mapping data has now been refined from sequence studies which indicate that gam encodes a protein of 174 amino acids Ci8.3 kDa~~th a startcodon 5801 bpfrom the LHE of Mu. (2) The gam protein protects linear DNA against exonuclease attack in vitro by virtue of being a DNA-binding protein. This binding is not specific for Mu DNA. Protection occurs with ds DNA that has flush ends or 3’ or 5’ 4-nt extensions, and does not require the presence of a 5’-terminal phosphate group.

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(3) Under appropriate conditions, pgum stimulates the frequency of transformation of linear plasmid DNA lo- to loo-fold, and this stimulation occurs whether the DNA has been linearised by the action of restriction enzymes that leave flush or complementary ends. Moreover in experiments with the plasmid pBR322, which possesses appropriate restriction sites within a TcR gene, it could be shown that a higher than normal proportion of TcR transformants were recovered. (4) Excess production of pgam expressed from the Ip, promoter decreases cell viability. ACKNOWLEDGEMENTS

We would like to thank Nicola Ford for typing the manuscript, Joy Chessell and Sylvia Brings for clean glassware and media, and Sheila Maynard-Smith for her comments on the manuscript.

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